Retrievable hydrogels for ovarian follicle transplantation and oocyte collection

Abstract

Cancer survivorship rates have drastically increased due to improved efficacy of oncologic treatments. Consequently, clinical concerns have shifted from solely focusing on survival to quality of life, with fertility preservation as an important consideration. Among fertility preservation strategies for female patients, ovarian tissue cryopreservation and subsequent reimplantation has been the only clinical option available to cancer survivors with cryopreserved tissue. However, follicle atresia after transplantation and risk of reintroducing malignant cells have prevented this procedure from becoming widely adopted in clinics. Herein, we investigated the encapsulation of ovarian follicles in alginate hydrogels that isolate the graft from the host, yet allows for maturation after transplantation at a heterotopic (i.e., subcutaneous) site, a process we termed in vivo follicle maturation. Survival of multiple follicle populations was confirmed via histology, with the notable development of the antral follicles. Collected oocytes (63%) exhibited polar body extrusion and were fertilized by intracytoplasmic sperm injection and standard in vitro fertilization procedures. Successfully fertilized oocytes developed to the pronucleus (14%), two-cell (36%), and four-cell (7%) stages. Furthermore, ovarian follicles cotransplanted with metastatic breast cancer cells within the hydrogels allowed for retrieval of the follicles, and no mice developed tumors after removal of the implant, confirming that the hydrogel prevented seeding of disease within the host. Collectively, these findings demonstrate a viable option for safe use of potentially cancer-laden ovarian donor tissue for in vivo follicle maturation within a retrievable hydrogel and subsequent oocyte collection. Ultimately, this technology may provide novel options to preserve fertility for young female patients with cancer.

Keywords: alginate; biomaterial; follicle; hydrogel; oocyte.

Figure 1. Follicle Survival and Growth in Hydrogel Explants 7 Days Post-Transplantation in Subcutaneous and Bursa Sites

Antral, multi-layered secondary, and secondary follicles were observed in extracted ovarian grafts from the subcutaneous and bursa sites (A–B, C–D). In panel A, surrounding alginate material served to maintain separation of host tissue from follicles transplanted subcutaneously. A representative antral follicle is denoted with an asterisk (*) in panels A and C. Large numbers of primordial and primary follicles were identified in all extracted hydrogels and denoted with a single (*) or double asterisk (**) respectively in panels B and D. Hydrogel implant in panel A–B and C–D contained approximately 360 and 1,100 follicles, respectively. Similar results were observed for alginate hydrogels with follicle populations of ~730 ovarian follicles at both sites. Scale bar: 100 μm.

Figure 2. Percentage of Follicle Populations Recovered from Alginate Hydrogel Explants 7 Days Post-Transplant

Follicle populations (primordial, primary, secondary, multi-layered secondary, and antral) were quantified and displayed as percent of recovered follicles for the 1100, 730, and 360 follicle implant conditions for the (A) subcutaneous (n=3/follicle condition) and (B) bursa transplant sites (n=4 for 1,100 follicle and 730 follicle condition, n=3 for 360 follicle condition). (C) Percentage of surviving follicles for the subcutaneous and bursa transplant sites (±SEM).

Figure 3. Egg Retrieval from Encapsulated Follicles in Alginate Hydrogels in Subcutaneous Site 7 Days Post-Transplant and Their Meiotic Maturation In Vitro

(A) Follicles were easily identified in explanted hydrogels. (B) MII oocytes were confirmed via polar body extrusion (indicated with black arrows). (C) Egg retrieval and MII status from the subcutaneous site. Of the GV oocytes collected from antral follicles in Day 7 explants with 1,100 ovarian follicles, 34/54 oocytes were MII post-in vitro maturation (63% MII rate) from 3 trials. Oocytes collected from Day 7 explants with 730 or 360 ovarian follicles resulted in an MII rate of 55% (6 MII oocytes/11 GV oocytes) and 46% (10 MII oocytes/22 GV oocytes collected), respectively (D) Normal spindle morphology (indicated by an arrow) was also confirmed in subcutaneously-matured oocytes. Image (D) depicts an oocyte obtained from an explant containing ovarian follicles from 3 ovaries. Scale bar: 100 μm (A, B).

Figure 4. Egg Retrieval from Encapsulated Follicles in Alginate Hydrogels in Bursa Site 7 Day Post-Transplant and Their Meiotic Maturation In Vitro

(A) Germinal vesicle (GV) oocytes were retrieved from antral follicles (B) MII oocytes were confirmed via polar body extrusion (indicated with black arrows) (C) Egg retrieval and MII status from the bursa site. Of the 15 GV oocytes collected at Day 7 from 1 trial, 3 were MII post-IVM (20% MII rate). Note: MII follicles were only observed with the 1,100 follicle condition and not for the 730 or 360 follicle condition. (D) Normal spindle morphology (indicated by an arrow) was also confirmed in MII oocytes. Image (D) depicts an oocyte obtained from an explant containing ovarian follicles from 3 ovaries. Scale bar: 100 μm (A, B).

Figure 5. Fertilization Competency of MII Oocytes Matured in Subcutaneous Site is Assessed via Intracytoplasmic Sperm Injection (ICSI) and In Vitro Fertilization (IVF)

(A) Post-ICSI, MII oocytes progressed to the pronucleus, 2-cell, and 4-cell embryonic stages. Post-IVF, denuded eggs progressed to the pronucleus and 2-cell stages. Scale bar: 100 μm (B) After 14 MII oocytes were injected with sperm via ICSI, embryos progressed to the pronucleus (14%), 2-cell (36%), and 4-cell (7%) stages, while 43% of oocytes remained in MII arrest (C) Embryos resulting from 10 MII oocytes denuded and placed in an IVF dish with sperm, progressed to the pronucleus (20%) and 2-cell (40%) stages, while 40% of oocytes remained in MII arrest. Results obtained from 2 ICSI trials and 1 IVF trial.

Figure 6. In Vivo Imaging of NSG Mice Pre- and Post-Removal of Cancer-Laden Alginate Hydrogels

(A) Representative image of NSG mouse with subcutaneously-transplanted alginate hydrogel containing 200 MDA-MB-231 cells expressing luciferase (imaged Day 7 post-transplant). The presence of cancer cells in the hydrogels was confirmed by the positive luminescent signal (signal detection is 600–60,000 counts). The hydrogel implant at Day 7 is denoted with a black arrow. For the control group, mice transplanted with only ovarian follicles were imaged. Cancer cells were present only in the gel implant. (B) Mice were imaged 3 weeks after hydrogels removal to assess cancer cell presence. Cancer cells were not detected in experimental mice that had cancer-laden hydrogels removed at Day 7. Cancer cells were not detected in negative controls as well. 360 ovarian follicles were also incorporated into transplanted alginate hydrogels. n=4 per group.

Figure 7. Absence of Metastatic Lesions in Liver and Lungs of Recipient Mice 3 Weeks Post-Removal of Hydrogel

Representative image of (A) liver from naïve NSG control (n=3), (B) lung from naïve NSG control (n=3), (C) liver from recipient mouse with 200 cancer cells in hydrogel (n=4), and (D) lung from recipient mouse with 200 cancer cells in hydrogel (n=4). Lung and liver tissue removed at 3 weeks did not contain any cellular abnormalities or metastatic growths according to staining with hematoxylin and eosin (H&E). Scale bar: 100 μm.